Chloroplasts and flow cytometry

From: Patricex Petit (pxpetit@cochin.inserm.fr)
Date: Sat Jan 22 2000 - 06:26:41 EST


answer to

I've been approached by an investigator who wants to separate mesophyl
chloroplasts from bundle sheath chloroplasts in maize, and this is way out
of my area of expertise.  They gave me a reference with Bob Ashcroft's name
on it, so I was hoping to get some advice from the expert!  The papers were
fairly old and detailed several methods including the use of differences in
scatter and in fluorescence emission in the 685-710 range to isolate them.
They also used ratios of fluorescence above and below 710, which seems to
relate to the emission difference between chlorophylls A and B.  Is this
necessary (as we have no filter currently to do this) or is there another
way to do this?  Is anyone out there doing something similar?

Carol Oxford
Manager, UC Davis Optical Biology Lab
Tupper Hall, rm 3439
University of California
Davis, California  95616
(530) 752-7205
(530) 752-4548 fax
(530) 523-0967 pager

I have been publishing onthat subject a long time ago. I link somes
references below.

1. Schröder W.P. and Petit P.X. (1992) Flow cytometry of spinach
chloroplasts: determination of the intactness and lectin-binding properties
of the enbvelope and thylakoid membranes. Plant Physiol. 100 : 1092-1102.

2. Kausch, A.P., T.P. Owen, Jr., S. Narayanswami, and B.D. Bruce. 1999.
Organelle isolation by magnetic immunoabsorption. Biotechniques 26: 336-43.

3. Pfundel, E., and A. Meister. 1996. Flow cytometry of mesophyll and
bundle sheath chloroplast thylakoids of maize (Zea mays L.). Cytometry 23:
97-105.

4. Wolters, A.A., A.C. Vergunst, F. van der Werff, and M. Koornneef. 1993.
Analysis of nuclear and organellar DNA of somatic hybrid calli and plants
between Lycopersicon spp. and Nicotiana spp. Mol Gen Genet 241: 707-18.

In fact, the idea of separating mesophyll and bundle sheath chloroplasts
and mitochondria is an old idea...
Which was supposed to be an alternative to the clasical preparation and
purification on percoll gradients.
But is it realistic to do so?....

The purification of chloroplast of both origin in an very intact conditions
appears to me very problematic and the flow cytometry approach will make
you very desapointed because the technology is not fair in the sense that
she will tell you the exact quality of your preparation. Anyway, if you
find somes informations in the 1992 paper, just E-mail me and I will help
you as much as possible

Petit P.X. and Cantrel C. (1986) Mitochondria from Zea mays leaf tissues:
differentiation of carbon assimilation and photorespiratory activity
between mesophyll and bundle sheath cells. Physiol. Plant. 67 : 442-446.

Petit P.X. (1984) Mitochondria from the mesophyll cells of Zea mays leaves.
J. Plant Physiol. 116 : 351-364.

Sincerely yours

Patroce


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Dr. Petit Patrice X.
Institut Cochin de Génétique Moléculaire
INSERM U129 - CHU Cochin Port-Royal
24, rue du Faubourg Saint-Jacques
F-75014 Paris, France.

Tel: 33 01 44 41 24 11
Fax: 33 01 44 41 24 21
E-mail: pxpetit@icgm.cochin.inserm.fr



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